Depression affects more than 17% of the overall population, with prevalence in women twice that in men. Estrogen treatment has been shown to result in elevation of mood, and botanicals, in particular from black cohosh (Actaea racemosa; AR), and red clover (Trifolium pretense; TP), have been reported to manifest antiestrogenic, estrogenic, and serotonergic activity. Botanicals may be proven as alternatives for hormone replacement therapy (HRT) and selective estrogen receptor modulator (SERM) therapy, which is needed given the health risks that are being increasingly associated with these therapies. The data from clinical trials on botanicals testing the benefits in menopausal complaints, including depression, is ambiguous. Unfortunately, whereas estrogens and SERMs have been well studied in a wide variety of animal models, such studies are relatively lacking on botanicals. It is the objective of this project to obtain convincing animal model data on the antidepressant activity of botanicals and to elucidate the biochemical pathways involved, in order to determine a basis for clinical studies. The central hypothesis of this proposal is that the antidepressant activity of estrogen, phytoestrogens, TP and AR botanicals is mediated at least in part via NO/cGMP signalling in the hippocampus and amygdala. It is an objective of this proposal to test this hypothesis by comparison of estrogen, botanicals and NO mimetics in an animal behavioural model and by immunocytochemistry directed at selected protein biomarkers associated with signaling cascades. Observations on the location and elevation of levels of selected proteins in amygdala and hippocampus will identify changes characteristic of antidepressant activity. A long term goal of the project, is the development of an in vitro screen for identifying antidepressant botanicals.